Natural diversity of lactococci in γ-aminobutyric acid (GABA) production and genetic and phenotypic determinants.

BACKGROUND: γ-aminobutyric acid (GABA) is a bioactive compound produced by lactic acid bacteria (LAB). The diversity of GABA production in the Lactococcus genus is poorly understood. Genotypic and phenotypic approaches were therefore combined in this study to shed light on this diversity. A comparative genomic study was performed on the GAD-system genes (gadR, gadC and gadB) involved in GABA production in 36 lactococci including L. lactis and L. cremoris species. In addition, 132 Lactococcus strains were screened for GABA production in culture medium supplemented with 34 mM L-glutamic acid with or without NaCl (0.3 M). RESULTS: Comparative analysis of the nucleotide sequence alignments revealed the same genetic organization of the GAD system in all strains except one, which has an insertion sequence element (IS981) into the PgadCB promoter. This analysis also highlighted several deletions including a 3-bp deletion specific to the cremoris species located in the PgadR promoter, and a second 39-bp deletion specific to L. cremoris strains with a cremoris phenotype. Phenotypic analysis revealed that GABA production varied widely, but it was higher in L. lactis species than in L. cremoris, with an exceptional GABA production of up to 14 and 24 mM in two L. lactis strains. Moreover, adding chloride increased GABA production in some L. cremoris and L. lactis strains by a factor of up to 16 and GAD activity correlated well with GABA production. CONCLUSIONS: This genomic analysis unambiguously characterized the cremoris phenotype of L. cremoris species and modified GadB and GadR proteins explain why the corresponding strains do not produce GABA. Finally, we found that glutamate decarboxylase activity revealing GadB protein amount, varied widely between the strains and correlated well with GABA production both with and without chloride. As this protein level is associated to gene expression, the regulation of GAD gene expression was identified as a major contributor to this diversity.

Glutamate-induced metabolic changes in Lactococcus lactis NCDO 2118 during GABA production: combined transcriptomic and proteomic analysis.

GABA is a molecule of increasing nutraceutical interest due to its modulatory activity on the central nervous system and smooth muscle relaxation. Potentially probiotic bacteria can produce it by glutamate decarboxylation, but nothing is known about the physiological modifications occurring at the microbial level during GABA production. In the present investigation, a GABA-producing Lactococcus lactis strain grown in a medium supplemented with or without glutamate was studied using a combined transcriptome/proteome analysis. A tenfold increase in GABA production in the glutamate medium was observed only during the stationary phase and at low pH. About 30 genes and/or proteins were shown to be differentially expressed in glutamate-stimulated conditions as compared to control conditions, and the modulation exerted by glutamate on entire metabolic pathways was highlighted by the complementary nature of transcriptomics and proteomics. Most glutamate-induced responses consisted in under-expression of metabolic pathways, with the exception of glycolysis where either over- or under-expression of specific genes was observed. The energy-producing arginine deiminase pathway, the ATPase, and also some stress proteins were down-regulated, suggesting that glutamate is not only an alternative means to get energy, but also a protective agent against stress for the strain studied.

Influence of feeding and analytical method on the bioequivalence of a racemic drug undergoing enantioselective enterohepatic recycling.

Two crossover bioequivalence trials of an enantioselectively enterohepatic recycled drug, carprofen, were conducted in dogs with the same racemic oral formulation to determine: (i) the influence of feeding patterns, and (ii) the effect of the analytical method (enantioselective vs non-enantioselective) on the statistical power of the trials. The first trial was conducted with a standard feeding protocol and the second with a special feeding protocol selected to ensure constant biliary flow into the duodenum. Using a non-enantioselective technique, 90% confidence intervals provided conclusions of bioequivalence in 100% of the cases for both area under the plasma concentration versus time curve (AUC) and maximum plasma drug concentration (Cmax) with the special feeding protocol, but only 50% for AUC and 13% for Cmax with the standard feeding protocol, suggesting that a feeding pattern that diminishes plasma drug concentration rebound for an enterohepatically recycled drug increases the power of a bioequivalence trial. Whatever the feeding protocol, an enantioselective method decreased the power of the trials for AUC but increased the power of the trials for Cmax. For an enterohepatically recycled drug, feeding pattern can influence the power of a bioequivalence trial, and the analytical technique that provides the greatest power depends on the assessed bioequivalence parameter and the feeding pattern.